The genus Trichoderma is a well-known cosmopolitan soil fungus that has been widely explored as an antagonist of numerous plant pathogenic fungi (Howell C R, 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease 87:4-10; Harman G E, 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96:190-194). Isolates of Trichoderma species can be successful in plant disease control due to directly antagonizing pathogen activity and/or inducing host resistance responses (Harman G E, 2000. Myths and dogmas of biocontrol: Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Disease 84: 377-393). Furthermore, Trichoderma's function as a plant growth promoter has been reported for some strains after establishment as a non-strict plant symbiont by colonizing the rhizosphere (Harman G E, Howell C R, Viterbo A, Chet I, Lorito M, 2004. Trichoderma species—Opportunistic, avirulent plant symbionts. Nature Reviews Microbiology 2:43-56; Harman G E, Kubicek P K, 1998. Trichoderma and Gliocladium Vol 2. Enzymes, biological control and commercial applications. Taylor and Francis, London 1-393). Different modes of action for Trichoderma strains employed as biocontrol agents were described: a) rhizosphere competence by colonizing the soil and/or parts of the plant or by competition for nutrients; b) mycoparasitism by producing a wide variety of cell wall degrading enzymes against pathogens; c) antibiosis via production of antimicrobial compounds (volatiles and non-volatiles) that can kill the pathogens; d) growth promotion by improving plant development and e) induction of systemic defensive responses in plants (Harman and Kubicek, 1998, ibid; Harman, 2006, ibid).
Currently, the majority of Trichoderma products in the biopesticide marketplace are based on solid substrate-produced aerial conidia. Aerial conidia of Trichoderma are produced using solid substrate fermentation on moistened grains and this process takes weeks for production and drying, which consequently increases the production costs (Pandey A, Fernandes M, Larroche C, 2008. Current developments in solid-state fermentation. Springer New York, US, 517p. DOI: 10.1007/978-0-387-75213-6; Ramanujam B, Prasad R D, Rangeswaran R, 2010. Mass production, formulation, quality control and delivery of Trichoderma for plant disease management. The Journal of Plant Protection Sciences 2(2): 1-8). The production of fungal conidia on moistened grains suffers from numerous constraints including high labor costs, poor quality control, long fermentation times, environmental concerns for workers, and difficulties in scale-up. Liquid culture production methods have been investigated and focused on the production of submerged conidia and chlamydospores of Trichoderma (Lewis J A, Papavizas G C, 1983. Production of chlamydospores and conidia by Trichoderma spp. in liquid and solid growth media. Soil Biology and Biochemistry 15: 351-357; Papavizas G C, Dunn M T, Lewis J A, Beagle-Ristaino J, 1984. Liquid fermentation technology for experimental production of biocontrol fungi. Phytopathology 74(10): 1171-1175; Tabachnik M, 1989. Method of growing Trichoderma. U.S. Pat. No. 4,837,155A; Harman G E, Jin X, Stasz T E, Peruzzotti G, Leopold A C, Taylor A G, 1991. Production of conidial biomass of Trichoderma harzianum for biological control. Biological Control 1: 23-28; Jin X, Taylor A G, Harman G E, 1996. Development of media and automated liquid fermentation methods to produce desiccation-tolerant propagules of Trichoderma harzianum. Biological Control 7: 267-274; Sriram S, Roopa K P, Savitha M J, 2011. Extended shelf-life of liquid fermentation derived talc formulations of Trichoderma harzianum with the addition of glycerol in the production medium. Crop Protection 30:1334-13339). Formulation studies focused on stabilization processes for Trichoderma biomass, aerial conidia and chlamydospores that provided adequate storage stability (Lewis J A, Papavizas G C, 1985. Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil. Plant Pathology 34(4): 571-577; Jin X, Custis D, 2010. Microencapsuling aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures. Biological Control 56: 202-208; Yonsel Y S, Batum M S, 2010. Trichoderma granule production. Patent EP20080866322; Sriram et al., 2011, ibid). Despite these attempts to produce Trichoderma in liquid culture, low yields, long fermentation times and poor desiccation tolerance and storage stability have impaired the large-scale adoption of this production methodology by industry.
To meet the biopesticide market expectations and promote Trichoderma's use as a fungicide or to promote plant health, an efficient and feasible liquid culture production technology must be developed to conceive a high quality Trichoderma-based product. Preferably, the Trichoderma would be persistence in soil and decaying plant material. To that end, many plant pathogenic fungi produce sclerotia; i.e., melanized, compact hyphal aggregates that are highly resistant to desiccation. These propagules often serve as the overwintering structure for the fungus (Cooke, 1983, Morphogenesis of sclerotia. In “Fungal Differentiation: A Contemporary Synthesis” Smith, J. E, ed. pp 397-418. Marcel Dekker, Inc., New York, N.Y., U.S.A.; Coley-Smith and Cooke, 1971, Survival and germination of fungal sclerotia. In “Annual Review of Phytopathology”, Horsfall, J. G., Baker, K. F., Zentmyer, G. A., eds. pp 65-92. Annual Reviews Inc., Palo Alto, Calif., U.S.A.). Microsclerotia (small sclerotial particles, 200-600 um) of fungal plant pathogens such as Colletotrichum truncatum and Mycoleptodiscus terrestris have been produced in high concentration in submerged liquid culture fermentation (Jackson and Schisler, 1995, Mycological Research, 99:879-884; Shearer and Jackson, 2003, U.S. Pat. No. 6,569,807). Microsclerotia of these pathogens of weedy plants have shown value as persistent propagules in soil and aquatic environments (Shearer and Jackson, 2006, Biological Control. 38:298-306; Boyette et al., 2007, BioControl 52:413-426). However, to date, microsclerotia have not been reported for any Trichoderma species.